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1.
William Veatch 《Journal of cellular biochemistry》1976,5(4):431-451
Gramicidin A is a linear polypeptide antibiotic that facilitates the diffusion of monovalent cations across lipid bilayer membranes by forming channels. It has been proposed that the conducting channel is a dimer which is in equilibrium with nonconducting monomers in the membrane. To directly test this model in several independent ways, we have prepared and purified a series of gramicidin C derivatives. All of these derivatives are fully active analogs of gramicidin A, and each derivative has a useful chromophore esterified to the phenolic hydroxyl of tyrosine #11. Simultaneous conductance and fluorescence measurements on planar lipid bi-layer membranes containing dansyl gramicidin C yielded four conclusions: (1) A plot of the logarithm of the membrane conductance versus the logarithm of the membrane fluorescence had a slope of 2.0 ± 0.3, over a concentration range for which nearly all the gramicidin was monomeric. Hence, the active channel is a dimer of the nonconducting species. (2) In a membrane in which nearly all of the gramicidin was dimeric, the number of channels was approximately equal to the number of dimers. Thus, most dimers are active channels and so it should be feasible to carry out spectroscopic studies of the conformation of the transmembrane channel. (3) The association constant for dimerization is more than 1,000-fold larger in a glycerolester membrane with 26 Å-hydrocarbon thickness than in a 47 Å-glycerolester membrane. The dimerization constant in a 48 Å-phosphatidyl choline membrane was 200 times larger than in a 47 Å-glycerolester membrane, showing that it depends on the type of lipid as well as on the thickness of the hydrocarbon core. (4) We were readily able to detect 10?14 mole cm?2 of dansyl gramicidin C in a bilayer membrane, which corresponds to 60 fluorescent molecules per square μm. The fluorescent techniques described here should be sufficiently sensitive for fluorescence studies of reconstituted gates and receptors in planar bilayer membranes. An alternative method of determining the number of molecules of gramicidin in the channel is to measure the fraction of hybrid channels present in a mixture of 2 chemically different gramicidins. The single-channel conductance of p-phenylazo-benzene-sulfonyl ester gramicidin C (PABS gramicidin C) was found to be 0.68 that of gramicidin A. In membranes containing a mixture of these 2 gramicidins, a hybrid channel was evident in addition to 2 pure channels. The hybrid channel conductance was 0.82 that of gramicidin A. Fluorescence energy transfer from dansyl gramicidin C to diethylamino-phenylazobenzene-sulfonyl ester gramicidin C (DPBS gramicidin C), provided an independent way to measure the fraction of hybrid channels on liposomes. For both techniques the fraction of hybrid channels was found to be 2ad where a2 and d2 were the fractions of the 2 kinds of pure channels. This result strongly supports a dimer channel and the hybrid data excludes the possibility of a tetramer channel. The study of hybrid species by conductance and fluorescence techniques should be generally useful in elucidating the subunit structure of oligomeric assemblies in membranes. The various models which have been proposed for the conformation of the gramicidin transmembrane channel are briefly discussed. 相似文献
2.
Sheila Galt John Sandblom Yngve Hamnerius Per Hjevik Elisabeth Saalman Bengt Nordn 《Bioelectromagnetics》1993,14(4):315-327
The hypothesis that specific combinations of DC and low frequency AC magnetic fields at so-called cyclotron-resonance conditions could affect the transport of ions through ion channels, or alter the kinetics of ion channels (opening and closing rates), has been tested. As a model system, the ion channels formed by gramicidin A incorporated in lipid bilayer membranes were studied. No significant changes in channel conductance, average lifetime, or formation rate as a function of applied fields could be detected over a wide range of frequencies and field strengths. Experiments were carried out to measure the time-resolved single-channel events and the average conductances of many-channel events in the presence of K+ and H+ ions. The channel blocking effect of Ca++ was also studied. © 1993 Wiley-Liss. Inc. 相似文献
3.
Single-channel conductance fluctuations are analysed for gramicidin A incorporated into binary-mixed black lipid membranes of charged phosphatidic acid and neutral lecithin in different molar ratios. At very low Ca++ concentrations in the electrolyte (i.e. in the presence of EDTA) homogeneous lipid mixtures are identified through their conductance and life time probability distributions for integral gramicidin pores. As for the pure lipid components, the conductance histograms each show a single maximum with regular width and for all channels a single mean lifetime is found.For Ca++-levels (10-6–10-5
M) that are close to the critical demixing concentration (10-4
M) unusually broad conductance distributions and reduced lifetimes are found provided the PC content, x, of the membrane is close to the critical mixture (x
crit0.5). We interpret this as a first example of the coupling of a membrane function (the transport of ions) to a lipid matrix with locally fluctuating composition close to a critical demixing point.For
the conductance histogram of gramicidin A in an equimolar mixture of PA and PC shows two well-separated maxima. A correlation analysis between conductance and lifetime of the single pores shows that the two channel populations also differ significantly in their mean channel lifetime, *. This finding is interpreted as being direct evidence for Ca++-induced lateral phase separation in black lipid membranes, as has been postulated recently.Abbreviations used HEPES
N-2-hydroxyethyl-piperazine-N-2-ethane-sulfonic acid
- EDTA
ethylenediaminetetraacetic acid 相似文献
4.
We have previously characterized the “RCA” channel (root Ca2+ channel), a voltage-dependent, Ca2+-permeable channel found in plasma membrane-enriched vesicles from wheat roots incorporated into artificial planar lipid bilayers.
Earlier work indicated that this channel was insensitive to 1,4-dihydropyridines (DHPs, such as nifedipine and 202–791). However,
the present study shows that this channel is sensitive to DHPs, but only with submillimolar Ca2+, when the probability of channel opening is reduced, with flickery closures becoming increasingly evident as Ca2+ activity decreases. Under these ionic conditions, addition of nanomolar concentrations of (+) 202–791 or nifedipine caused
an increase in both the probability of channel opening and the unitary conductance. It is proposed that there is a competitive
interaction between Ca2+ and DHPs at one of the Ca2+-binding sites involved in Ca2+ permeation and that binding of a DHP to one of the Ca2+-permeation sites facilitates movement of other calcium ions through the channel. The present study shows that higher plant
Ca2+-permeable channels can be greatly affected by very low concentrations of DHPs and that channel sensitivity may vary with
the ionic conditions of the experiment. The results also indicate interesting structural and functional differences between
plant and animal Ca2+-permeable channels. 相似文献
5.
Erwin Neher 《生物化学与生物物理学报:生物膜》1975,401(3):540-544
The thallous ion was found to interact very specifically with gramicidin channels in black lipid membranes. Although the ion itself carries current through the channel better than Na+ or K+, it blocks Na+ currents at concentrations which are two orders of magnitude lower than the Na+ concentrations. 相似文献
6.
In phosphatidylserine membranes the decrease in the conductance of the gramicidin A single channel caused by calcium is attributed to a reduction of surface potential and to a direct blocking of the pore (Apell et al. 1979).The aim of this paper is to make a, quantitative evaluation of these two effects. We recorded the conductance of gramicidin single channels in 100 mM KCl in the presence of different amounts of CaCl2, MgCl2 or TEACl.The ionic activities at the channel mouth were calculated using the Gouy-Chapman-Stern theory. Our experiments showed that even when the K+ activity at the channel mouth was estimated to be the same, the single channel conductance was lower if divalent cations were present. This effect is attributed to a blocking action of these ions.Abbreviations PS
phosphatidylserine
- TEA
tetraethylammonium 相似文献
7.
M Str?ssle G Stark M Wilhelm 《International journal of radiation biology and related studies in physics, chemistry, and medicine》1987,51(2):265-286
The ion channel formed by the pentadecapeptide gramicidin A in planar lipid membranes is extremely sensitive to ionizing radiation. The membrane conductance may drop by several orders of magnitude under appropriate experimental conditions (low pH and presence of oxygen). The radiation sensitivity is strongly reduced for gramicidin M-. This analogue has the four tryptophan residues replaced by phenylalanines. Experiments performed in the presence of various radical scavengers suggest that the inactivation of the channel is due to a combined action of OH and of HO2 radicals at the tryptophan residues. The shape of the inactivation curves following continuous radiolysis or pulse radiolysis were found to be in fair agreement with a simple model which assumes that the damage of a single tryptophan residue is sufficient for channel inactivation. The conductance of inactivated channels could not be resolved within the experimental accuracy. This is contrary to photolysis of gramicidin channels found by Busath and Waldbilling (1983), where a broad distribution of low conductance states was observed. The inactivation by radiolysis seems to represent an 'all-or-none-process' of the channel conductance. 相似文献
8.
Gramicidin A, a linear peptide antibiotic, makes membranes permeable to alkali cations and hydrogen ions by forming transmembrane channels. We report here conductance and fluorescence energy transfer studies of channels containing two kinds of gramicidin. These studies of hybrid channels were designed to determine the number of molecules in a channel. The gramicidins studied were gramicidin A, dansyl gramicidin C, the p-phenylazobenzene sulfonyl derivative of gramicidin C (PABS4 gramicidin C), and the 4-(diethylamino)-phenylazobenzene-4-sulfonyl chloride derivative of gramicidin C (DPBS gramicidin C). The dansyl, PABS and DPBS groups were linked to the hydroxyl group of tyrosine 11 in gramicidin C. The single-channel conductance of PABS gramicidin C in planar bilayer membranes is 0.68 that of gramicidin A. Membranes containing both PABS gramicidin C and gramicidin A exhibit three kinds of channels: a pure gramicidin A, a pure PABS gramicidin C channel, and a hybrid channel with an intermediate conductance (0.82 that of gramicidin A). The dependence of the frequencies of these three kinds of channels on the mole fractions of gramicidin A and PABS gramicidin C in the membrane-forming solution fits a dimer model. Fluorescence energy transfer was used as a complementary means of ascertaining the frequency of hybrid channels. Dansyl gramicidin C was the fluorescent energy donor and DPBS gramicidin C was the energy acceptor. The efficiency of energy transfer between these chromophores in hybrid channels in liposomes was 75%. The relative quantum yield of the dansyl fluorescence was measured as a function of the mole fraction of DPBS gramicidin C. These fluorescence studies, like the single-channel conductance measurements, showed that there are two molecules of gramicidin in a channel. The study of hybrid species by conductance and fluorescence techniques should be generally useful in elucidating the subunit structure of oligomeric assemblies in membranes. 相似文献
9.
The conductance induced by gramicidin A in lipid bilayer membranes has been shown to be made up of discrete, well-defined units. In 0.1 M NaCl, and for 100 mV applied, the integral conductance of the unit channel at 20 °C is 5.8·10−12 Ω−1. 相似文献
10.
If the ion concentration is low enough that most channels are unoccupied, then the ‘independence relations’ should be satisfied and the permeability ratio should equal the conductance ratio. It has been previously reported that for the gramicidin A channel these ratios for Na+ and K+ were not equal at concentrations as low as 10 mM. However, these ratios were not measured at the same applied potential, as is required by the theory. Instead, the conductance ratio was measured at 100 mV and corrected using calculated current-voltage relations. In this report the comparison between permeability and conductance ratios is reexamined using data obtained at the correct potential. There is no significant difference in the ratios at 10 mM when they are measured at the same voltage. This implies that most channels are not occupied by sodium or potassium ions at 10 mM. 相似文献
11.
Sergei V. Sychev Stanislav V. Sukhanov Leonid I. Barsukov Vadim T. Ivanov 《Journal of peptide science》1996,2(3):141-156
The relation between the various spatial structures of the gramicidin A channels and their ionic conductance has been studied. For this aim, various conformations of the peptide were pre-formed in liposomal bilayer and after subsequent fusion of liposomes with planar lipid bilayer the measured channel conductance was correlated with gramicidin structures established in liposomes. To form the single-stranded π6.3π 6.3 helix the peptide and lipid were co-dissolved in TFE prior to liposome preparation. THF and other solvents were used to form parallel (↑ ↑ π π) and antiparallel (↑ ↓ π π) double helices. Conformation of gramicidin in liposomes made by various phosphatidylcholines was monitored by CD spectroscopy, and computer analysis of the spectra obtained was performed. After fusion of gramicidin containing liposomes with planar bilayer membranes from asolectin, the histograms of single-channel conductance were obtained. The histograms had one or three distinct peaks depending on the liposome preparation. Assignment of the structure of the channel to conductance levels was made by correlation of CD data with conductance histograms. The channel-forming analogue, des(Trp-Leu)2-gramicidin A, has been studied by the same protocol. The channel conductances of gramicidin A and the shortened analogue increase in the following order: ↑ ↓ π π 2 ↑ ↑ π π < π 6.3π6.3. Single-channels formed by double helices have higher dispersity of conductance than the π6.3π6.3 helical channel. Lifetimes of the double helical and the π6.3π6.3 helical channels are very close to each other. The data obtained were compared with theoretically predicted properties of double helices [1]. 相似文献
12.
The N-terminally truncated derivative of salmon calcitonin (sCt) (acetyl-[Asn30,Tyr32]-calcitonin fragment 8-32) (AC 187) lacks hormonal activity and is a potent and selective antagonist of the hormone and amylin
receptor. It was investigated for its capability to interact and form channels in palmitoleoylphosphatidylcholine:dioleoylphosphatidylglycerol
planar lipid membranes. Interestingly, AC 187 exhibits channel activity, whose parameters, i.e., central conductance (Λ
c), occurrence (number of channels/min), voltage-dependence and lifetime, are similar to those found for sCt although, in the
same experimental conditions, it takes longer to incorporate into the membrane than sCt. This channel activity can be modulated
by changing either the holding potential or the pH of the medium, or by adding picomolar concentrations of SDS. One evident
difference between the two peptides is that sCt is unselective (1.03) while AC 187 displays a cationic selectivity (P
K
+/P
Cl
− = 2.7) at pH 7, increasing to 3.87 when the pH drops to 3.8. The present findings indicate that the 1-7 disulfide bridge
is sufficient but not necessary for membrane interaction, in accordance with the observation reported on the interaction with
membrane receptors. Furthermore, the remarkable pH dependence of the cationic channel could be taken into consideration for
full biotechnological study.
Proceedings of the XVIII Congress of the Italian Society of Pure and Applied Biophysics (SIBPA), Palermo, Sicily, September
2006. 相似文献
13.
Hool LC 《European biophysics journal : EBJ》2005,34(5):369-376
The first ion channels demonstrated to be sensitive to changes in oxygen tension were K+ channels in glomus cells of the carotid body. Since then a number of hypoxia-sensitive ion channels have been identified. However, not all K+ channels respond to hypoxia alike. This has raised some debate about how cells detect changes in oxygen tension. Because ion channels respond rapidly to hypoxia it has been proposed that the channel is itself an oxygen sensor. However, channel function can also be modified by thiol reducing and oxidizing agents, implicating reactive oxygen species as signals in hypoxic events. Cardiac ion channels can also be modified by hypoxia and redox agents. The rapid and slow components of the delayed rectifier K+ channel are differentially regulated by hypoxia and -adrenergic receptor stimulation. Mutations in the genes that encode the subunits for the channel are associated with Long QT syndrome and sudden cardiac death. The implications with respect to effects of hypoxia on the channel and triggering of cardiac arrhythmia will be discussed. 相似文献
14.
The equivalence of fluctuation analysis and chemical relaxation measurements: a kinetic study of ion pore formation in thin lipid membranes 总被引:6,自引:0,他引:6
(1) Autocorrelation measurements were made of the current fluctuations due to ion-conducting channels produced by gramicidin A in black lipid membranes. (2) Relaxation experiments using a voltage jump were made on the same system. (3) Reciprocal time constants were determined by both methods over a 108 fold range in membrane conductivity. starting from the single channel level. (4) The mean squared amplitudes of the fluctuations were determined from the autocorrelation functions. (5) The data were tentatively rationalized on the basis of a hypothetical dimerization reaction, assuming that gramicidin A dimers form conducting channels. The same forward rate constants are obtained, via (3), by both methods (1) and (2). The backward rate constant agrees excellently with direct measurements of the mean life time of a conducting channel. (6) The unit channel conductance and - assuming again a dimerization - the equilibrium constant can be obtained from the fluctuation amplitude distribution. 相似文献
15.
The action of metal polycations and pH on ionic channels produced in bilayer lipid membranes (BLM) by three different toxins was studied by measuring membrane capacitance and channel conductance. Here, we show that critical concentrations of Cd2+, La3+ or Tb3+ induce complex changes in membrane capacitance. The time course of capacitance changes is similar to the time course of channel blocking by these ions at low concentration. No changes in BLM capacitance or conductance were observed in the range of pH 5.8–9.0. A pH shift from 7.4 to 3–4 or 11–12 induced large changes in BLM capacitance and channel conductance. For all studied channel-forming proteins, the initial capacitance increase preceded the conductance decrease caused by addition of polycations or by a change in pH. A close relationship between membrane lipid packing and ion channel protein is suggested. 相似文献
16.
The channel forming properties of synthetic gramicidin A and dLeu2-gramicidin A were compared in black lipid membranes. The most probable single channel conductance was identical for both derivatives but in each case a distribution of smaller channel sizes was observed. However, the lifetime of the channel formed by dLeu2-gramicidin A was considerably shorter than for gramicidin A. The dLeu2 substitution is considered to interfere with the head to head hydrogen bonding which forms the conducting dimer, thus destabilizing the dimeric structure of the channel and reducing the lifetime. This represents the first demonstration of side-chain modulation of channel lifetime. 相似文献
17.
Summary According to the model of Urry, the cation-permeable gramicidin channel is a dimeric helix formed by association of two peptide monomers linked at their amino ends. In this paper the channel properties of gramicidin analogs are described which have been obtained by chemical modification at the coupling site of the two half-channels. In these analogs the amino terminal-CHO group is replaced by-CO(CH2)
n
COOH(n=2, 3, 4, 5, 6). All analogs form conducting channels in black lipid membranes with the same general properties as found for gramicidin A. The observation that the channel-forming activity decreases with increasing pH is consistent with the notion that the half-channels are linked at the amino terminus. The channel lifetime of the different analogs varies between 2 msec and 50 sec, the longest lifetime being found for the compound withn=3. The single-channel conductance is always smaller than that of gramicidin A, but the reduction of depends on the nature of the permeable ion. Ion specificity was studied at 1m electrolyte by measuring the conductance for different permeable ions (Na+, K+, Cs+). The conductance ratio(Cs+)/(Na+) was found to vary between 2 and 10.5 for the different analogs. 相似文献
18.
The effects of heating, on an aqueous gramicidin A lysolecithin system, were examined by carbon-13 nuclear magnetic resonance (13C-NMR), circular dichroism (CD), and sodium-23 nuclear magnetic resonance (23Na-NMR), and the results are collectively interpreted to indicate micellar-packaging of gramicidin channels and cation occupancy in the channel. 13C-NMR of the gramicidin-lysolecithin system demonstrates a decrease in mobility of the micellar lipid on heating which is indicative of incorporation of gramicidin into the hydrophobic core of the micelle. A unique and reproducible CD spectrum is obtained for the heat incorporated state. Sodium-23 spin-lattice relaxation times (T1) demonstrated sodium interaction to be dependent on heat incorporation. The T1 identified interaction is blocked by silver ion which is known to block sodium transport through the channel in lipid bilayer studies. The temperature dependence of the sodium-23 line width defines an exchange process with an activation energy of 6.8 kcal/mole which is essentially the same as the activation energy reported for transport through the channel in lecithin bilayer studies, and the sodium exchange process is blocked by thallium ion which is also known to block sodium transport through the channel. 相似文献
19.
Yuri N. Antonenko Tatyana I. Rokitskaya Arthur J. L. Cooper Boris F. Krasnikov 《Journal of bioenergetics and biomembranes》2010,42(2):151-163
Minocycline (an anti-inflammatory drug approved by the FDA) has been reported to be effective in mouse models of amyotrophic
lateral sclerosis and Huntington disease. It has been suggested that the beneficial effects of minocycline are related to
its ability to influence mitochondrial functioning. We tested the hypothesis that minocycline directly inhibits the Ca2+-induced permeability transition in rat liver mitochondria. Our data show that minocycline does not directly inhibit the mitochondrial
permeability transition. However, minocycline has multiple effects on mitochondrial functioning. First, this drug chelates
Ca2+ ions. Secondly, minocycline, in a Ca2+-dependent manner, binds to mitochondrial membranes. Thirdly, minocycline decreases the proton-motive force by forming ion
channels in the inner mitochondrial membrane. Channel formation was confirmed with two bilayer lipid membrane models. We show
that minocycline, in the presence of Ca2+, induces selective permeability for small ions. We suggest that the beneficial action of minocycline is related to the Ca2+-dependent partial uncoupling of mitochondria, which indirectly prevents induction of the mitochondrial permeability transition. 相似文献
20.
Summary Dielectric permittivities have been determined for suspensions of lysolecithin packaged malonyl gramicidin channels over the frequency range of 5kHz to 900 MHz and under conditions of approximately equimolar concentrations (10mM) of channels and salts. The salts were lithium chloride, sodium chloride and thallium acetate. A relaxation process unique to the thallium acetate-channel system was observed which on analysis gave rise to a relaxation time at 250 of 120 nsec. The permittivity data, as well as a comparison of binding constants, indicate that the relaxation process results from Tl+ being bound within the channel and more specifically from an intrachannel ion translocation with a rate constant of approximately 4×106 sec–1 and with an energy of activation of less than 6.7 kcal/mole. These data compare favorably with data from conductance studies on planar bilayers and with ion and carbon-13 nuclear magnetic studies on the lysolecithin packaged malonyl gramicidin channels which combine to indicate that the relaxation process is due to the jump of the thallium ion across a central barrier. 相似文献